Chapter 8 Flashcards
Diabetes mellitus is
a disease caused by a deficiency in the production or activity of the hormone insulin (a protein), resulting in elevated blood glucose levels.
- about 5 % of all deaths r by diabetes.
- can lead to many health complications, including blindness, organ problems, limb amputations, & early death.
Type 1 diabetes
- a type of diabetes caused by an inability to produce insulin
- Many with type 1 diabetes need insulin by mouth or by injection to prevent the disease from seriously
damaging their body
Type 2 diabetes
- a type of diabetes caused by low insulin or an inability to use insulin
- 2 main risk factors of type 2 diabetes r a genetic predisposition & being overweight.
- North America = type 2 diabetes is rising –> Canadians eat more refined foods & sugar, & less natural foods & fibre, than ever before.
- more people r suffering from insulin resistance, obesity, and type 2 diabetes.
Insulin was isolated by which 2 researchers?
Dr. Frederick Banting and Dr. Charles Best, at the University of Toronto in 1922
- Helen Free, born a year after the discovery, invented a method to analyze the blood sugar by dip-test urinalysis. –> (Before this, doctors tasted a patient’s urine. Sweet urine was an indicator of diabetes.)
-allowed people with diabetes to monitor their blood sugar level easily at home. These two innovations have vastly improved the lives of diabetics
Insulin was initially collected from the pancreases of ________ & __________. What changed?
pigs, cows
- was allergic reactions to this insulin, even though it was necessary to keep them alive.
- Scientists tried to mass-produce acc human insulin.
- They discovered that they could insert the human insulin gene into bacteria, & the bacteria would make human insulin.
Genetic engineering is
the intentional production of new genes and alteration
of genomes by the substitution or introduction of new genetic material
Bacteria are versatile tools for genetic engineers cuz..
1) they reproduce quickly & often,
2) are relatively inexpensive to maintain
3) contain plasmids
What is used to make biosynthetic human insulin?
- Escherichia coli (AKA E.coli) is used.
- this bacteria is very common in the human body
- E. coli transcribes & translates the piece of human DNA to make insulin, which is then harvested from the bacteria.
- Safflowers r also used to make insulin.
- After human insulin gene in the plasmid is inserted into the E. coli or the safflower, both organisms have recombinant DNA
recombinant DNA is
a DNA strand that is created using DNA pieces from two or more sources (does it have to be species?)
Restriction Enzymes
-1st step in genetic recombination is to isolate, or cut out, a DNA fragment that contains the desired gene
- Restriction enzymes (AKA endonucleases) occur naturally in prokaryote cells & cut DNA at specific locations
–> they act as molecular scissors
- Each enzyme recognizes a unique nucleotide sequence and cuts at one specific location & direction. –> Each restriction enzyme recognizes a specific sequence of nucleotides on a DNA strand = recognition site for that particular enzyme
- When the enzyme cuts the DNA molecule, the pieces it makes r known as restriction fragments.
Each restriction enzyme _____________ at only 1 ______________ and in only 1 ____________. Give an example.
cuts (or digests), recognition site, direction
- enzyme EcoRI binds to a recognition site with the base-pair sequence 5’-GAATTC-3’ and cuts the DNA backbone between G and A.
-Notice that the recognition sites on the DNA are palindromic when you consider both strands
-Another EcoRI enzyme makes the same cut in the complementary DNA strand. - This leaves only a small # of the H-bonds holding the DNA molecule together, allowing the DNA molecule to be easily separated, resulting in complementary “sticky” ends.
A restriction enzyme is
- AKA restriction endonuclease
an enzyme that cuts DNA at a specific location in a
base sequence
A recognition site is
a sequence of bases on a DNA strand that restriction enzymes bind to.
A restriction fragment is
a fragment that is produced when a DNA strand is cut by a restriction enzyme
- Restriction enzymes r highly specific.–> Most have recognition sites for 4 to 8 base pairs.
–> The fewer base pairs that they need for the recognition sequence the more cuts that are made in a DNA strand.
–> In a random DNA sequence, the probability of
finding a particular four-pair sequence is 1 in 4^4
( 1/256 or 0.4 %).
–> The chance of finding the six-pair sequence that EcoRI needs to perform a cut is 1 in 4^6 (1/4096 or 0.02 %).
2 possible outcomes result from a restriction enzyme cutting a DNA molecule.
- If cuts r made straight across the strand, blunt ends r created.
- If cuts are made in a zigzag, sticky ends r created.
- Ex EcoRI produces sticky ends, whereas SmaI
produces blunt ends - biologists prefer restriction enzymes that produce sticky ends cuz the DNA fragments that r created r easier to join to any other DNA strand that has been cut by the same enzyme.
A blunt end is
the end that remains after restriction enzymes cut straight across a DNA strand; a blunt end is more difficult than a sticky end to recombine with another strand
A sticky end is
the end that remains after restriction enzymes cut on a zigzag across a DNA strand; a sticky end of a DNA
fragment can form hydrogen bonds with a complementary sticky end on any other DNA molecule that has been cut by the same enzyme
Discovery of Restriction Enzymes
Discovered accidentally in 1970 by Dr. Hamilton Smith at Johns Hopkins University when he was studying how bacteria resisted viral infections
- Since then, Over 2500 restriction enzymes identified, with specificity for ~200 target sequences.
~200 restriction enzymes are commercially available for molecular biology applications.
What is the Biological Function of restriction enzymes?
- Protect bacteria by cutting viral DNA or RNA injected during infection.
- When a virus injects its own DNA or RNA into a bacterial cell, the bacterial restriction enzymes destroy the viral nucleic acid by cutting it in pieces
How is a restriction enzyme named?
- is named after its cell of origin, plus a Roman numeral if more than one restriction enzyme has been isolated from this species.
- Ex, EcoRI (pronounced “eco-R-one”) was the first restriction enzyme isolated from E. coli,
- XhoI was the first enzyme isolated from Xanthomonas holcicola,
- HindII & HindIII were the 2nd & 3rd enzymes isolated from Haemophilus influenzae
DNA Ligase is
is the enzyme that is used to join cut strands of DNA
- Works best with sticky ends but T4 DNA ligase is effective with blunt ends.
- The 2 DNA fragments must have overlapping complementary portions or be blunt ends that r properly aligned end to end
- Fragments are complementary if generated by the same restriction enzyme. –> Hydrogen bonds form between the complementary bases, but this is not a stable arrangement.
- The DNA is not fully linked until phosphodiester bonds form between the backbones of the double strands.–> DNA ligase makes this happen.
- DNA ligase facilitates a dehydration reaction, releasing H2O as phosphodiester bonds form –> Fully links DNA strands to create stable double-stranded DNA.
Plasmids
- 2nd tool needed for recombinant DNA techniques
- are small circular pieces of DNA that r found in bacteria
- replicate independently of the chromosomal DNA.
- often contain genes that code for specific proteins, like those that provide resistance to antibiotics such as ampicillin or protect from the toxic effects of certain heavy metals.
- In fact, when u hear about bacteria that mutate quickly so that diseases become difficult to treat, it is often the plasmid DNA that is mutating.
A competent cell is
a cell that is able to take up foreign (often plasmid) DNA from its surroundings
- Ex, E.coli
A vector is
a DNA molecule that is used as a vehicle to transfer foreign genetic material into a cell. Ex, a plasmid
The plasmid copy number is
the # of plasmids of a specific type within a cell
- is variable & is characteristic of a particular plasmid.
- If a plasmid with a high copy # has been engineered to produce insulin, more insulin will be produced per cell cuz more copies of the insulin-producing gene are present
How are plasmids attached to DNA gene fragments to make recombinant DNA?
- If the target gene fragment and a plasmid are cut with the same restriction enzyme, they will have complementary sticky ends.
- The foreign fragments and the plasmid fragments can be placed in the same solution, where they anneal cuz of the complementary sticky ends.
- DNA ligase is then added to re-form the phosphodiester bonds between the fragments, resulting in a new circular piece of DNA that carries the foreign gene fragment. –> plasmid now = recombinant DNA
- The recombinant plasmid is introduced into a host cell (commonly bacteria). –> The plasmid replicates within the host cell, making multiple identical copies of the gene (gene cloning).
- The gene will now begin to express its function. Ex, if the insulin gene is in the plasmid, the host cell will begin to produce insulin
A restriction map is
a diagram that shows the relative locations of all known restriction enzyme recognition sites on a particular plasmid & the distances, in base pairs (bp), between the sites.
- when u add the bp up, we can see the plasmid’s total length in bp
- cuz plasmids r circular, the # of fragments is always equal to the # of cuts
- allows molecular biologists to determine which plasmids & restriction enzymes might be most suitable for a particular recombo DNA procedure & to evaluate quickly the success of the cloning experiments
Plasmids: Transformation
- Plasmids can enter bacterial cells, replicate, and express inserted foreign genes.
- The successful introduction of DNA from another source is = transformation
- The cell that has received the DNA is said to be transformed
If a bacterial cell is not able to take up a plasmid that contains foreign DNA, it can sometimes be made competent in a laboratory. ELABORATE
1 is to place the bacteria in a solution that contains CaCl2 & recombinant plasmids in an
ice-water bath.
- As the bacteria cool, the calcium ions stabilize the negative phosphate ions on the phospholipid bilayer of the cell membrane.
- The CaCl2 solution is then heated quickly & re-cooled.–> sudden change from cold to hot momentarily disrupts the membrane, allowing the plasmid to enter.
- The cells r then kept at 37 °C for a period of time to stabilize & grow.
-After the cells have stabilized & grown, they r tested for ampicillin resistance. Those with a plasmid with ampicillin resistance gene grow in a medium that contains ampicillin. This means that they were successfully made competent & transformed
DNA hybridization
- technique of DNA hybridization is used to identify the cells that contain the introduced plasmids with the desired gene
- This gene can be identified by its unique DNA sequence cuz it will pair with a short, single-stranded complementary DNA molecule, called a hybridization probe.
- For insulin production, the probe matches a DNA segment coding for insulin (15–2500 bases long).
- A series of steps taken to identify a target DNA sequence during DNA hybridization.–> After the presence of the DNA for insulin production is confirmed, the bacteria r grown in huge quantities,
enough to produce insulin on a commercial scale.
Lake Sturgeon
- Lake sturgeon were once abundant in Canadian lakes but r now endangered due to overfishing for their meat & caviar.
- It is illegal to fish or consume lake sturgeon under the federal Fisheries Act.
- If conservation officers come across campers eating fish, & they suspect that the fish is not in season or is an endangered species, they can take a small sample to a forensic lab.
- If the DNA matches that of a banned species, the conservation officers have sufficient evidence to give to the police.
Trent University, in Ontario, has a Wildlife DNA Profiling & Forensics Laboratory
- lab tracks endangered species & supports the enforcement of the Convention on International Trade in Endangered Species (CITES).
- The polymerase chain reaction (PCR), developed in 1983 by Kary Mullis, helps them by amplifying small DNA samples for extensive testing.
- DNA fingerprinting identifies species from minimal samples, such as fish on a plate or blood on a hunting knife.
- PCR and DNA fingerprinting help analyze DNA to determine the species source, enforce conservation laws, and provide forensic evidence.
The Polymerase Chain Reaction is
- AKA PCR
a process that is used to make a huge # of copies of a DNA sequence in a laboratory, quickly (in a few hours) & without a host organism - It is a simple, reliable, fast, and inexpensive method widely used in molecular biology.
- Kary Mullis, who developed PCR, was awarded the Nobel Prize in Chemistry in 1993.
- PCR focuses on replicating a specific DNA region, not the entire genome.
- The process occurs outside a cell’s nucleus, in a laboratory setting.
- The whole process takes place in a microfuge tube
(a small test tube)
PCR consists of three steps:
1) denaturation
2) annealing
3) elongation
- These steps r repeated in many cycles, usually 30-40
PCR: Denaturation
- 1st step of PCR
- a double-stranded DNA molecule is denatured, or separated, into its 2 single strands –> occurs when the DNA strand is heated to 94 to 96 °C for 20 to 40 s, breaking the H-bonds holding the 2 strands together.
PCR: Annealing
- 2nd step of PCR
- occurs at a lower temperature (50–65 °C) for 20–40 seconds
- Single-stranded DNA primers anneal to complementary sequences on the separated DNA strands.
- 2 primers r used, each complementary to opposite strands near the 3’ ends of the target sequence. –> This results in both primers being oriented in the 5’ to 3’ direction toward the target sequence.
- The specific nucleic acid sequences of the primers ensure that only the target DNA fragment is amplified. –> This specificity makes PCR a highly accurate and reliable technique.
A DNA primer is
a short single-stranded DNA sequence, easily made in a lab, that is complementary to a sequence at 1 end of the target sequence
